Chemosynthetic cyclo-hepta modified peptide capable of inhibiting toxin of staphylococcus aureus and use thereof

ABSTRACT

The invention relates to a chemosynthetic cyclo-hepta modified peptide capable of inhibiting toxins of  Staphylococcus aureus  and a use thereof. The chemosynthetic peptide can specifically inhibit production of the toxins of  Staphylococcus aureus  by means of the binding of the RNAIII activator protein by a  Staphylococcus aureus  autocrine. The invention also relates to the use of the chemosynthetic peptide in the pharmaceutical field. The sequence general formula of the chemosynthetic cyclo-hepta modified peptide is CH 3 —(CH 2 )m-X-G-(C-Q-H—W—W—H—W—Y—C)—(R)n-Y. The results show that the modified peptide can be dissolved well in water and has good activity against the toxins of  Staphylococcus aureus.

FIELD OF THE INVENTION

The present invention relates to a chemosynthetic cyclo-hepta modifiedpeptide capable of inhibiting the toxins of Staphylococcus aureus and ause thereof. The chemosynthetic peptide can specifically inhibitproduction of the toxins of Staphylococcus aureus by means of thebinding of the RNAIII activator protein by a Staphylococcus aureusautocrine. The invention also relates to the use of the chemosyntheticpeptide in the pharmaceutical field.

BACKGROUND OF THE INVENTION

Staphylococcus aureus is a common gram-positive pathogen, which is oneof the main microorganisms that cause fatal diseases such as burn andwar wound infection, pneumonia, endocarditis, septicemia, toxic shockand so on. The number of people infected with Staphylococcus aureus inhospitals only exceeds millions a year. At present, the treatment ofStaphylococcus aureus is mostly combination of antibiotics, but theeffect is not satisfactory. Because Staphylococcus aureus is susceptibleto drug resistance, which has no good solution, many common antibioticsare ineffective to it. Controlling Staphylococcus aureus infection isone of the problems to be solved in clinical medicine.

The main pathogenic substances of Staphylococcus aureus are toxinsincluding hemolytic toxins, leukocides and enterotoxins. The lasteststudies show that the synthesis of these virulence factors inStaphylococcus aureus is controlled by an adjustable RNA molecule andRNAIII. RNAIII activates the gene transcription of virulence factors andregulates the translation of virulence factors through basecomplementation. In the early logarithmic growth phase of bacteria, thelevel of RNAIII is low, but in the late logarithmic phase growth phase,the level of RNAIII increases by 40 times. The level of RNAIII isregulated by the protein secreted by Staphylococcus aureus itself andRANIII activating protein (RAP), so the factor RAP is also calledStaphylococcus aureus virulence stimulator. Staphylococcus aureussecretes RAP continuously and activates virulence factor only after RAPreaches a certain concentration. Staphylococcus aureus itself is notpathogenic without RAP. In 1998, the research result published byBalaban et al. in the journal Science show that by immunizing animalswith RAP prepared, the antibody can effectively protect mice from theinfection with Staphylococcus aureus (Balaban N, et al. Autoinducer ofvirulence as a target for vaccine and therapy against Staphylococcusaureus. Science, 1998, 280(17):438-440). Using phage display technique,the inventor screened the small molecular cyclo-heptapeptide MRK(CQHWWHWYC), which can specifically bind to RAP molecule and inhibit itsactivity, from the random cyclo-heptapeptide library, please refer toCN0315020.5 and other previous work. In vitro and in vivo experimentsshow that the small peptide may inhibit the production of the toxins ofStaphylococcus aureus. However, in the subsequent studies, the inventorfound that the small peptide has a major defect-insoluble in water orphysiological solution, only soluble in organic solvents (such as DMSO),which seriously affects its bioactivity and makes the small peptideunusable in clinic. For this reason, this has become a major problemtroubling inventor, so that research and development work cannot beadvanced. After repeated screening experiments, the inventor triedvarious structural modification methods, including the introduction ofhydrophilic groups such as PEG, nanocrystallization by nanotechnology,and even the use of liposome or micelle drug loading, but these methodshave little effect, even greatly affect the activity and stability ofcyclo-heptapeptide. In addition, in recent years, In the field ofenzyme, protein and peptide pharmaceuticals, there have been a number ofreports on chemical modifications, such as improved stability andlong-lasting effects (2012-2013 Development Report on Biochemistry andMolecular Biology, China Science and Technology Press, 2014.04), butwhat kind of chemical modification is useful remains complicated due todifferent function and structure, and needs to be determined accordingto specific situation.

SUMMARY OF THE INVENTION

The invention relates to the chemical modification ofcyclo-heptapeptide, belonging to a small molecular peptide. The mainpurpose of the invention is to improve water solubility and maintainbiological activity. The small peptide is chemically modified on thebasis of the cyclo-heptapeptide and the sequence general formula isCH₃—(CH₂)m-X-G-(C-Q-H—W—W—H—W—Y—C)—(R)n-Y. The results show thatchemosynthetic cyclo-hepta modified peptide (abbreviated as MRG) notonly can be dissolved well in water, but also has good activity againstthe toxins of Staphylococcus aureus compared with its originalcyclo-heptapeptide, so that the above-mentioned technical problems aresolved. Terminology: cyclo-heptapeptide refers to C-Q-H—W—W—H—W—Y—C,abbreviated as MRK.

The compound of the present invention is completely new in both sourceand structure, and has not been reported in any literature.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a novel chemosynthetic cyclo-hepta modifiedpeptide capable of inhibiting the toxins of Staphylococcus aureus and apreparation method thereof. The chemosynthetic peptide can specificallyinhibit the production of the toxins of Staphylococcus aureus.

Another purpose of the invention is to provide the use of thechemosynthetic peptide in the pharmaceutical field.

The compound of the present invention is a small molecular polypeptideanalogue (hereinafter referred to as “the peptide”, “cyclo-heptamodified peptide”) having the following general chemical structuralformula:

CH₃—(CH₂)m-X-G-(C-Q-H—W—W—H—W—Y—C)—(R)n-Y;

wherein,

m=0-20, preferably 3-17, more preferably 6-14, most preferably 8-12;

X is selected from a group consisting of CONH, NHCO, O or S, preferablyX=CONH;

n=1-10, preferably 1-7, more preferably 2-4, most preferably 3;

Y is selected from OH or NH₂;

G represents natural L-glycine residues or D-type isomers thereof;

C represents natural L-cysteine residues or D-isomers thereof, Qrepresents natural L-type glutamine residues or D-type isomers thereof,H represents histidine residues or D-type isomers thereof, W representsnatural L-type tryptophan residues or D-type isomers thereof, Yrepresents natural L-tyrosine residues or D-type isomers thereof, Rrepresents natural L-type arginine residues or D-type isomers thereof,and two cysteines represented by C are linked by a disulfide bond.

The peptide can specifically bind to the virulence stimulator RAP ofStaphylococcus aureus and inhibit the production of the toxins ofStaphylococcus aureus.

The peptide is synthesized by chemical synthesis. In other words, thepeptide is obtained by chemical synthesis.

wherein CH₃—(CH₂)m- represents the alkyl portion of the alkanoyl group,preferably CH₃(CH₂)₁₀— (m=10), which forms a lauroyl modified G whenbeing linked to X=CONH.

G, C, Q, H, W, Y, R independently represent the following groups: Grepresents natural L-glycine residues or D-type isomers thereof; Crepresents natural L-cysteine residues or D-type isomers thereof; Qrepresents natural L-glutamine residues or D-isomers thereof; Hrepresents histidine residues or D-type isomers thereof; W representsnatural L-tryptophan residues or D-type isomers thereof; Y representsnatural L-tyrosine residues or D-type isomers thereof; R representsnatural L-arginine residues or D-isomers thereof.

The two cysteines of the 9 amino acid sequences C-Q-H—W—W—H—W—Y—C arelinked by a disulfide bond.

A solid-phase synthesis method for preparing the peptide comprises thefollowing steps:

Step 1, the conventional protected amino acids are coupled one by onewith Rink Amide-AM Resin as the starting carrier, and the substitutiondegree sub is 0.45-0.55 mmol/g.

Step 2, under the action of the condensation reagents of1-hydroxybenzotriazole (HOBT) and N, N′-diisopropyl carbodiimide (DIC),by using a solid-phase polypeptides stepwise condensation method, theamino acids protected by Fmoc are coupled one by one according to thesequence of the peptides from C to N terminals to obtain:

Gly-Cys(Trt)-Gln(Trt)-His(Trt)-D-Trp(Boc)-Trp(Boc)-His(Trt)-Trp(Boc)-Tyr(tBu)-Cys(Trt)-Arg(pbf)-Arg(pbf)-Arg(pbf)-Rink Amide-AM Resin.

Step 3, under the action of the condensation reagent ofO-benzotriazole-N,N,N′,N-tetramethyl-uronium-hexafluorophosphate (HBTU)and N,N-diisopropyl ethylamine (DIPEA), dodecanoic acid is coupled toobtain:CH₃(CH₂)₁₀CO-Gly-Cys(Trt)-Gln(Trt)-His(Trt)-D-Trp(Boc)-Trp(Boc)-His(Trt)-Trp(Boc)-Tyr(tBu)-Cys(Trt)-Arg(pbf)-Arg(pbf)-Arg(pbf)-RinkAmide-AM Resin.

Step 4, a conventional cracking reagent is mixed with the linear peptideresin obtained in step 3 to be subjected to cracking reaction, so as toremove Rink Amid-AM Resin and MRG side chain protection group, and thenthe MRG linear crude peptide is obtained by sedimentation,centrifugation, washing and drying.

Step 5, the linear crude peptide obtained in step 4 is grinded topowder, and the MRG linear crude peptide is dissolved in a mixedsolution of pure water and acetonitrile (2:1) until the concentration is1 mmol/500 mL, thus obtaining a linear crude peptide solution.

Step 6, diluted ammonia is dropwisely added to the MRG linear crudepeptide solution to adjust the pH of the MRG linear crude peptidesolution to 7.1-7.3, and stirred at a temperature of about 30° C. to besubjected to cyclization experiment. The cyclization time is 20-40 min.Whether the cyclization is complete is detected by high performanceliquid chromatography (HPLC) and the cyclization is terminated by addingacetic acid to adjust pH to the acidic condition after the cyclizationis complete.

Step 7, the target product crude product solution of step 6 is separatedand purified by C₁₈ reversed-phase high performance liquidchromatography column, and the high purity product is obtained afterrotary evaporation and freeze-drying.

The invention also relates to the use of the peptide in the preparationof drugs against Staphylococcus aureus infection.

The chemosynthetic peptide with the above structure can specificallybind to the virulence stimulator RAP of Staphylococcus aureus andinhibit its activity.

The invention particularly relates to the peptide improving thesolubility of the “cyclo-heptapeptide” (C-Q-H—W—W—H—W—Y—C).

The main reason of resistance to antibiotics in traditional antibioticstherapy is that bacteria produce an inducible enzyme that decomposeseffective groups of antibiotics under the pressure of survival. Theinvention utilizes the polypeptide compound specifically inhibiting RAPactivity to establish a treatment scheme for Staphylococcus aureusinfection, and finds a new outlet for the treatment of the common,recurrent and fatal drug-resistant Staphylococcus aureus infection whichhas been troubling the clinic.

The invention is of great significance to the development of novel smallmolecule peptide medicines resistant to Staphylococcus aureus infection,and has wide use value and broad market prospect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: HPLC purity identification chromatogram of chemosyntheticcyclo-heptapeptide MRK

FIG. 2: Mass spectrum identification chromatogram of chemicalsynthesized cyclo-heptapeptide MRK

FIG. 3: HPLC purity identification chromatogram of chemosyntheticcyclo-hepta modified peptide MRG

FIG. 4: Mass spectrometric identification chromatogram of chemosyntheticcyclo-hepta modified peptide MRG

FIG. 5: Therapeutic effect of the chemosynthetic peptide on bacteremiainduced by Staphylococcus aureus infection in mice

EXAMPLES

The present invention is further described in detail by examples below.

Example 1: Preparation of Chemosynthetic Cyclo-Heptapeptide MRK by SolidPhase Synthesis 1. Synthetic Peptide Sequence: CQHWWHWHWYC (End-to-EndRing Forming, Molecular Weight 1346.5) 2. Synthesis Steps:

Step 1, raw materials needed for solid state synthesis

H-Cys(Trt)-2-Chlorotrityl Resin(Shanghai Jier Biochemistry),Fmoc-Tyr(tBu)-OH, Fmoc-Trp-OH, Fmoc-His(Trt)-OH, Fmoc-Gln-OH,Fmoc-Cys(Trt)-OH, DCC, HOBt, TFAEDT, m-Cresol.

Step 2, solid state synthesis

Standard Fmoc-AA-OH/DCC/HOBt method

1.1 g H-Cys(Trt)-2-Chlorotrityl Resin (0.6 mmol) is used as the carrier,both amino acids and condensation agents are 3 times excessive, and2.218 gCys(Trt)-Gln-His(Trt)-Trp-Trp-His(Trt)-Trp-Tyr(tBu)-Cys(Trt)-2-ChlorotritylResin is obtained after synthesis.

Step 3, peptide resin cracking

210 mg crude product linear peptide is obtained with 0.5 gCys(Trt)-Gln-His(Trt)-Trp-Trp-His(Trt)-Trp-Tyr(tBu)-Cys(Trt)-2-ChlorotritylResin, 0.1 mL m-Cresol, 0.3 mL EDT, 7.5 mL TFA reacting at 0° C. for 90min.

Step 4, linear peptide oxidation

200 mg crude linear peptide is dissolved in 2000 mL water, pH isadjusted to 7-8 with NaHCO₃, K₃Fe(CN)₆ (2 mg/mL) solution is slowlydropwisely added to the reaction solution to show light yellow, and thenthe reaction continues for 1 hour. Cyclic peptide 76.7 mg (purity >95%)is obtained by solid phase extraction with C₁₈ column and elution with80% acetonitrile solution.

Step 5, purification and structure confirmation

RP-HPLC purification is carried out with C₁₈ column, the purity of theproduct is greater than 99% (FIG. 1). Structural confirmation isperformed by mass spectrometry and amino acid composition analysis, witha molecular weight of 1346.5 (FIG. 2).

Example 2: Preparation of Chemosynthetic Cyclo-Hepta Modified PeptideMRG by Solid-Phase Synthesis

1. Synthetic Peptide Sequence: CH₃(CH₂)₁₀CO-G-(CQHwWHWYC)-RRR-NH₂(Molecular Weight 2055.08)

2. Synthesis Steps:

Step 1, Synthesis of 2 mmol MRG. Rink Amide-AM Resin is selected, andthe substitution degree is 0.45 mmol/g. According to the formula, theamount of blank resin=the molar amount of synthesized MRG/thesubstitution degree of blank resin, that is, the amount of blank resinRink Amide-AM Resin needed to be weighed=2 mmol/0.45 mmol/g=4.5 g. Theweighed blank resin is put into the reaction column and swollen forabout 30 min in a mixed solution of N, N-dimethyl formamide anddichloromethane (DMF/DCM=2/1), and then washed with N,N-dimethylformamide (DMF) as washing solution for 3 times.

Step 2, the protective group Fmoc is removed from the washed resin byde-protection solution, specifically comprising: 20% of thede-protection solution (Vpiperidin/VDMF=20%) is prepared and added tothe swollen blank resin in step 1, and then nitrogen is blown forreaction. The Fmoc protective group is removed at two times, 5 min and15 min respectively, ensuring the complete de-protection. After thede-protection is completed, indene detection reagent is used:

(a) anhydrous ethanol solution of 5% ninhydrin (w/v)(b) phenol: anhydrous ethanol solution (4:1, w/v)(c) pyridine

Two droplets of each test reagent are added, heated for 3 minutes at atemperature of 105° C. If the color is blue, the protective solution canbe pumped out. If no color is showed, de-protection is not complete, thereason should be found out immediately, and de-protection needs to becarried out again. Fmoc-Arg (pbf)-OH and condensation reagents1-hydroxybenzotriazole (HOBT) and N,N′-diisopropyl carbon diimine (DIC)are used for coupling reaction after washing with DMF for six times. Thereaction time is 2-3 h. After the reaction is finished, the colourdevelopment reaction experiment of ninhydrin is used to detect whetherthe reaction is complete, namely a small amount of resin is taken andtwo droplets of each of the three solutions are heated for 3 min at atemperature of 105° C. If the color is colorless, the reaction iscomplete. On the contrary, the reaction is incomplete, raw material issupplemented or repeatedly fed until the reaction is complete. The aminoacid in the peptide sequence is coupled at one time according to themethod described above to obtain:

Gly-Cys(Trt)-Gln(Trt)-His(Trt)-D-Trp(Boc)-Trp(Boc)-His(Trt)-Trp(Boc)-Tyr(tBu)-Cys(Trt)-Arg(pbf)-Arg(pbf)-Arg(pbf)-RinkAmide-AM Resin

Step 3, after the peptide sequence in step 2 is coupled to Gly, the lastgroup, namely dodecanoic acid (lauric acid), should be coupled when theprotective group is removed. It should be noted that dodecanoic acid(lauric acid) is coupled while the coupling, washing, deprotection anddetection time are the same. The condensation reagents arebenzotriazole-N,N,N′, N′-tetramethyl-uronium-hexafluorophosphate (HBTU)and N,N-diisopropyl ethylamine (DIPEA), the reaction time is about onehour. After the reaction is completed, the same method as above is usedto determine whether the reaction is complete. Under the condition ofcomplete reaction, the resin is washed with DMF for 3-4 times and theresin is shrunk with methanol (MeOH) for 2 times, shrinking at each timelasts for 10 min. Then the resin is vacuumized to quicksand state toobtain the linear peptide resin of the target peptide:

CH₃(CH₂)₁₀CO-Gly-Cys(Trt)-Gln(Trt)-His(Trt)-D-Trp(Boc)-Trp(Boc)-His(Trt)-Trp(Boc)-Tyr(tBu)-Cys(Trt)-Arg(pbf)-Arg(pbf)-Arg(pbf)-RinkAmide-AMResin

Step 4, 8.2 g quicksand peptide resin in step 3 is weighed. Firstly, 100mL conventional cracking reagent is prepared according to the ratio ofTFA:phenyl sulfide:EDT:anisole=90:5:3:2. 8-10 mL cracking reagent isadded for every 1 g peptide resin for cracking reaction after shakingwell, so that about 70 mL cracking reagent is taken from 100 mLconventional cracking reagent for cracking reaction, the reaction timeis about 2-3 h. 60 mL filtrate (partially being lost) is obtained byfiltering the resin after the reaction is completed. The filtrate isslowly added to anhydrous ether according to a sedimentation ratio offiltrate to anhydrous ether being 1:6, subjected to standing for 30 minand then settled fully, centrifuged, washed and dried to obtain linearcrude peptide:CH₃(CH₂)₁₀CO-Gly-Cys-Gln-His-D-Trp-Trp-His-Trp-Tyr-Arg-Cys-Arg-Arg-NH₂.After drying, the crude peptide is about 3.6 g.

Step 5, the linear crude peptide obtained in step 4 is grinded topowder, and the linear crude peptide of MRG is dissolved toconcentration of 1 mmol/500 mL with the mixed solution of pure water andacetonitrile (V water: V acetonitrile=2:1). The linear crude peptidesolution is 1000 mL, and the sample is analyzed by HPLC to locate theappearance time.

Step 6, diluted diluted ammonia is dropwisely added into the linearcrude peptide solution in step 5 to make the pH value of the solutionrange from 7.1 to 7.3 and then 30% hydrogen peroxide diluted by about 10times is slowly dropwisely added to the solution, wherein the ratio of30% hydrogen peroxide (usually the 30% hydrogen peroxide is dilutedbefore slowly adding) to the solution is 1 mmol:0.3 mL. After addinghydrogen peroxide, the temperature can be controlled at 30° C. forcyclization. In the process of cyclization, HPLC analysis is needed totrace whether the cyclization is complete or not, and whether thecyclization is carried out mainly according to the linear peptidedetected by HPLC analysis and the shifting of cyclized peptide. Thecyclization time is about 20-40 min. After the cyclization experiment iscompleted, the pH of acetic acid solution is adjusted to 4.5 and thecyclization is terminated. The objective crude product is obtained:

CH₃(CH₂)₁₀CO-Gly-Cyclo(Cys-Gln-His-D-Trp-Trp-His-Trp-Tyr-Cys)-Arg-Arg-Arg-NH₂.

Step 7, the objective crude peptide obtained in step 6 after completecyclization analyzed by HPLC is separated and purified by preparativeC₁₈ reversed-phase high performance liquid chromatography (HPLC). Thecrude peptide is subjected to rotary evaporation and freeze-drying toobtain fine product. The main steps are as follows:

The crude peptide solution after cyclization crude filtration isfiltered through 0.45 mum microporous membrane passing integrity test.After the filtration is completed, the solution is adjusted to theacidic pH for purification.

DIONEX U3000 high performance liquid chromatography is used for processof preparative HPLC separation and purification. An analytical columnC₁₈, 5 μm, 300 Å, 4.6*250 mm is used; the analysis conditions are asfollows: the mobile phase includes A phase of 1% TFA, and B phase ofacetonitrile. The preparative instrument is an innovative 5 cmpreparative HPLC; the preparative chromatographic column is C₁₈, 10 μm,100 Å, 150*250 mm reversed phase silica gel column.

The filtered crude solution is loaded by a preparative HPLC, injected ina 5 cm chromatographic column. The mobile phase includes: A phase is TFA(1 mL TFA is added to 1000 mL water), B phase is 100% acetonitrile.

Detection wavelength: lambda=230 nm; column temperature: roomtemperature; samples are collected by clean triangular flasks, thepurified solution with the purity of higher than 98% and any individualimpurity of less than 1% is qualified. Otherwise, the unqualified samplerepeats the above steps, the solution is completely processed, thequalified and unqualified classified purified solutions are obtained,and the above steps are repeated for secondary purification of theunqualified sample to get more qualified samples as far as possible, andthen rotary evaporation concentration is carried out.

Step 8, the subpackaged concentrated liquid is freeze-dried to obtainwhite powder, namely the finished product.

1) The concentrated sample solution is pre-frozen firstly, that is thesample is placed on the partition of freeze-drying box to be pre-frozen.The temperature of the product drops to −40 □ or below, and is kept forabout 120 min.

2) Sublimation drying is carried out: the temperature of electricheating is set to 0° C., deviation time is 1 min, the process is keptfor about 40 min.

The temperature of the electric heating is set to 10° C. and thedeviation time is 500 min.

The temperature of the electric heating is set to 35° C. and thedeviation time is 420 min.

3) Desorption: The temperature rises to about 33° C. and is kept forabout 240 min.

Step 9, purity identification and structure determination ofchemosynthetic cyclo-hepta modified peptide MRG.

The small peptide obtained by chemical synthesis is identified by HPLCand its purity is higher than 95% (FIG. 3). The molecular weight isidentified as 2055.08 (1028.54×2-2) by mass spectrometry (FIG. 4).

Example 3: Comparison of Water Solubility of ChemosyntheticCyclo-Heptapeptide MRK and Water Solubility of Cyclo-Hepta ModifiedPeptide MRG

Three parts of chemosynthetic cyclo-heptapeptide (MRK) are weighted, 1mg for each part, and added into 1 mL centrifugal tube, each part isadded with 1 mL water for injection, normal saline and dimethylsulfoxide (DMSO). The results show that chemosyntheticcyclo-heptapeptide (MRK) is insoluble in water or normal saline, andonly soluble in DMSO. The solubility of cyclo-heptapeptide MRK in waterfor injection or normal saline is less than 0.1 mg/mL. The same methodis used to detect cyclo-hepta modified peptide MRG. The results showthat cyclo-hepta modified peptide MRG is soluble in water for injection,normal saline and DMSO. Through calculation, the solubility of thecyclo-hepta modified peptide in water for injection or normal saline isgreater than 1 mg/mL. The results show that the water solubility ofcyclo-heptapeptide MRG is much better than that of cyclo-heptapeptideMRK.

In order to compare the difference of water solubility between thechemosynthetic cyclo-heptapeptide MRK and cyclo-hepta modified peptideMRG. The inventor scaled up the synthesis process and makes gradualtransition from small-scale to pilot-scale production, making thepreparation conditions more stable, and the prepared cyclo-heptamodified peptide MRG once again verifies the water solubility. Theresults show that the solubility of cyclo-heptapeptide MRK is less than0.1 mg/mL in water for injection or normal saline, but a greater amountof cyclo-hepta modified peptide MRG, specifically 10 mg, is mixed with 1mL water for injection or normal saline, all MRG is dissolved and noprecipitation is found by standing. The results of multiple experimentsare the same. Calculated in percent concentration, the solubility ofcyclo-hepta modified peptide MRG in water for injection or normal salineis greater than 1.0%, which completely solves the technical problem thatthe original cyclo-heptapeptide MRK is insoluble in water.

Example 4: Inhibitory Effect of Chemosynthetic Cyclo-Heptapeptide MRKand Cyclo-Hepta Modified Peptide MRG on the Production of the Toxins ofStaphylococcus aureus 1. Experimental Reagents, Consumables andInstruments

The chemosynthetic cyclo-heptapeptide MRK (CQHWWHWHWYC) and thechemosynthetic cyclo-hepta modified peptide MRG(CH₃(CH₂)₁₀CO-G-(CQHwWHWYC)-RRR-NH₂) are synthesized by ChinaTechPeptide (Suzhou) Co., Ltd. The purity is greater than 95%.Staphylococcus aureus 04018 strain is preserved by the Laboratory ofBiochemistry and Molecular Biology, Institute of Basic Medical Sciences,Academy of Military Medical Sciences. Blood plate (fresh blood agarculture board) is from Beijing Aoboxing Bio-tech Co. Ltd. BHI plate ismade by our laboratory. Imported BHI (Bacto™ Brain Heart Infusin) ispurchased from the BD Company, USA. DMEM medium is purchased from CIBCO,USA. Imported fetal cow serum is purchased from the PAN BIOTECH Company,USA. 0.22 μm membrane is purchased from PALL Corporation, USA. MDBKcells are preserved in the cell bank of our laboratory. Desktopcentrifuge is from EPPDORF Company, Germany. Enzyme linkage is fromMICROPLATE, USA. Cell culture bottles are purchased from CORNING, USA.Centrifugal tubes, graduated pipette, capillary burette and disposablesyringes and other consumables are from our laboratory.

2. Experimental Methods

For the determination method for inhibitory level on the toxins ofStaphylococcus aureus, please refer to literatures. (Yang G, et al. Anovel peptide screened by phage display can mimic TRAP antigen epitopeagainst Staphylococcus aureus infections. J Biol. Chem. 2005, 280:27431-27435), details are as follows:

1) Staphylococcus aureus strain 04018 frozen in refrigerator is scribbedto a blood plate or BHI plate; cultured in an incubator for 16 hoursuntil monoclonal colony is observed in the plate, and placed in a 4° C.refrigerator for standby use.

2) After 8 hours, the monoclonal colony in the plate is randomlyselected, 25 mL test tubes of the BHI medium is inoculated with themonoclonal colony; and then the two test tubes are shook in a shakingtable at a temperature of 37° C. at a speed of 200 rpm, the bacteria arecollected after 16 hours, the bacteria solution in the two test tubesare mixed evenly for standby use.

3) The chemosynthetic peptide MRK and MRG and the positive control (thetoxins of Staphylococcus aureus inhibitor protein TP) are dissolved innormal saline, diluted to different concentrations, and added tobacteria BHI medium. The final concentrations of small peptides are 0,1.5, 5, 15, 50, 150 and 500 μg/mL respectively. The solvent control isthe same amount of normal saline; the negative control is the BHI mediumof Staphylococcus aureus; and a normal cell control is also provided.

4) The chemosynthetic peptides of different concentrations areco-incubated with Staphylococcus aureus 04018 and cultured for 6 hours,and then bacteria are collected. 700 μL bacterial suspensions are addedto EP tubes, centrifuged at 8000 rpm for 5 min, supernatants areobtained, boiled at a temperature of 100° C. for 7 min, and centrifugedat a speed of 14000 rpm for 10 min. 10 μL supernatants are taken, addedinto a 96-microwell cell culture plate inoculated with 1×10<5>/mL MDBKcells. After continuous culture for 18-20 hours, 5 μL MTT solution (5mg/mL) is added to each microwell, 100 μL 10% SDS+0.01M HCl solution isadded to each microwell after 3 hours. After culture in the incubator ata temperature of 37° C. for 20 hours, detection at 595 nm and readingare carried out by an enzyme-linked immunometricmeter.

3. Experimental Results and Conclusions

The experimental results show that the chemosynthetic cyclo-heptamodified peptide MRG prepared by the example of the invention can bedissolved in the normal saline. The chemosynthetic peptide may inhibitthe production of the toxins of Staphylococcus aureus and has an obviousprotective effect on the proliferation inhibition of MDBK cells inducedby the toxins of Staphylococcus aureus, showing a certain dose-effectrelationship. The inhibitory effect of 5 μg/mL small peptide (g/mL) onthe production of the toxins of Staphylococcus aureus is greater thanthat of TP protein (positive control), but the chemosyntheticcyclo-heptapeptide MRK is insoluble in water, and its suspension couldnot inhibit the production of the toxins of Staphylococcus aureus (Table1 below is an observation on the inhibitory effect of chemosyntheticcyclo-heptapeptide MRK and cyclo-hepta modified peptide MRG on theproduction of the toxins of Staphylococcus aureus). This difference inantimicrobial activity is considered by the inventors to be the resultof solubility improvements and structural changes in cyclo-heptamodified peptide, rather than simple changes in solubility.

TABLE 1 Inhibitory effect of chemosynthetic peptide on production of thetoxins of Staphylococcus aureus Cell survival Group (dose) MDBK cell OD(M ± SD) (%) MRG (0.15 mg/mL) 0.473 ± 0.004 96 MRG (0.05 mg/mL) 0.454 ±0.015 92 MRG (0.005 mg/mL) 0.296 ± 0.001 60 MRK (0.15 mg/mL) 0.171 ±0.001 35 MRK (0.05 mg/mL) 0.098 ± 0.014 20 MRK (0.005 mg/mL) 0.123 ±0.011 25 TP positive control (1 mg/mL) 0.464 ± 0.028 94 normal salinecontrol 0.133 ± 0.008 27 Staphylococcus aureus 0.112 ± 0.030 23 negativecontrol BHI medium control 0.494 ± 0.017 100

Example 5: Therapeutic Effect of Chemosynthetic Cyclo-Hepta ModifiedPeptide MRG on Bacteremia Induced by Staphylococcus aureus Infection inMice 1. Experimental Reagents, Consumables and Instruments

The chemosynthetic cyclo-hepta modified peptide MRG(CH₃(CH₂)₁₀CO-G-(CQHwWHWYC)-RRR-NH₂) is synthesized by ChinaTech Peptide(Suzhou) Co., Ltd. The purity is greater than 95%. Staphylococcus aureusNewman strain is preserved by the Institute of Basic Medical Sciences,Academy of Military Medical Sciences. Blood plate (fresh blood agarculture board) is from Beijing Aoboxing Bio-tech Co., Ltd. BHI plate ismade by our laboratory. Imported BHI (Bacto™ Brain Heart Infusin) ispurchased from the BD Company, USA. Sodium pentobarbital is purchasedfrom Sigma Corporation, USA. The table top centrifuge is form EPPDORFCompany, Germany. Centrifugal tubes, graduated pipette, and disposablesyringes and other consumables are from our laboratory.

2. Experimental Methods

1) Preparation of Staphylococcus aureus Newman Solution

Newman monoclonal strain with hemolytic circles is selected, 3 mL BHImedium is inoculated with the strain, and the strain is shook overlightat a speed of 220 rpm and a temperature of 37° C.

1 mL overnight strain is centrifuged at a speed of 5000 rpm for 2 min,and the supernatant is removed.

20 mL aseptic PBS is added for resuspension at a speed of 5000 rpm for 5min, and the supernatant is removed.

5 mL aseptic PBS resuspended bacteria is diluted to OD600=0.2.

2) Establishment of Model of Bacteremia and Determination of PolypeptideActivity in BALB/c Mice

BALB/c mice (female, 8 weeks) are subjected to intraperitoneal injectionof anesthetic (1% pentobarbital) (200 μL for each mouse).

Newman solution (OD600=0.2, 100 μL for each mouse) is injected intoretrobulbar venous plexus.

After 30 minutes of infection, 6 mice are injected with polypeptide atenterocoelia (0.5 mg/mL, 200 μL for each mouse) and 7 mice in controlare injected with sterile water (200 μL for each mouse).

The times of the mouse in one week are recorded and used for drawingsurvival curves.

3. Experimental Results and Conclusions

The experimental results show that the chemosynthetic cyclo-heptamodified peptide MRG prepared by the example of the invention can reducethe death caused by bacteremia in mice induced by Staphylococcus aureusat a dose of 5 mg/kg body weight, and the survival rate is greatlyimproved compared with the control of aseptic water for injection. Thechemosynthetic cyclo-heptapeptide MRK also has a good inhibitory effecton Staphylococcus aureus infection in vivo (FIG. 5).

1. A peptide having the following general chemical structure formula:CH₃—(CH₂)m-X-G-(C-Q-H—W—W—H—W—Y—C)—(R)n-Y; wherein, m=0-20; X isselected from a group consisting of CONH, NHCO, O or S; n=1-10; Y isselected from OH or NH₂; G represents natural L-glycine residues orD-type isomers thereof; C represents natural L-cysteine residues orD-type isomers thereof, Q represents natural L-glutamine residues orD-type isomers thereof, H represents histidine residues or D-typeisomers thereof, W represents natural L-type tryptophan residues orD-type isomers thereof, Y represents natural L-tyrosine residues orD-type isomers thereof, R represents natural L-type arginine residues orD-type isomers thereof, and two cysteines represented by C are linked bya disulfide bond.
 2. The peptide of claim 1, wherein the peptide canspecifically bind to the virulence stimulator RAP of Staphylococcusaureus and inhibit the production of the toxins of Staphylococcusaureus; the peptide is synthesized by chemical synthesis.
 3. The peptideof claim 1, wherein CH₃—(CH₂)m- represents the alkyl portion of thealkanoyl group.
 4. The peptide of claim 1, wherein G, C, Q, H, W, Y, Rindependently represent the following groups: G represents naturalL-glycine residues or D-type isomers thereof; C represents naturalL-cysteine residues or D-type isomers thereof; Q represents naturalL-glutamine residues or D-isomers thereof; H represents histidineresidues or D-type isomers thereof; W represents natural L-tryptophanresidues or D-type isomers thereof; Y represents natural L-tyrosineresidues or D-type isomers thereof; and R represents natural L-arginineresidues or D-isomers thereof.
 5. The peptide of claim 1, wherein thetwo cysteines of the amino acid sequences C-Q-H—W—W—H—W—Y—C are linkedby a disulfide bond.
 6. The peptide of claim 1, wherein the peptide isobtained by chemical synthesis.
 7. A solid-phase synthesis method forpreparing the peptide of claim 1, wherein comprising the followingsteps: Step 1, the conventional protected amino acids are coupled one byone with Rink Amide-AM Resin as the starting carrier, and thesubstitution degree sub is 0.45-0.55 mmol/g; Step 2, under the action ofthe condensation reagents of 1-hydroxybenzotriazole (HOBT) andN,N′-diisopropyl carbodiimide (DIC), by using a solid-phase polypeptidesstepwise condensation method, the amino acids protected by Fmoc arecoupled one by one according to the sequence of the peptides from C to Nterminals to obtain:Gly-Cys(Trt)-Gln(Trt)-His(Trt)-D-Trp(Boc)-Trp(Boc)-His(Trt)-Trp(Boc)-Tyr(tBu)-Cys(Trt)-Arg(pbf)-Arg(pbf)-Arg(pbf)-RinkAmide-AM Resin Step 3, under the action of the condensation reagents ofbenzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluorophosphate (HBTU)and N,N-diisopropyl ethylamine (DIPEA), dodecanoic acid is coupled toobtain:CH₃(CH₂)₁₀CO-Gly-Cys(Trt)-Gln(Trt)-His(Trt)-D-Trp(Boc)-Trp(Boc)-His(Trt)-Trp(Boc)-Tyr(tBu)-Cys(Trt)-Arg(pbf)-Arg(pbf)-Arg(pbf)-RinkAmide-AM Resin; Step 4, a conventional cracking reagent is mixed withthe linear peptide resin obtained in step 3 to be subjected to crackingreaction, so as to remove Rink Amid-AM Resin and MRG side chainprotection group, and then the MRG linear crude peptide is obtained bysedimentation, centrifugation, washing and drying; Step 5, the linearcrude peptide obtained in step 4 is grinded to powder, and the MRGlinear crude peptide is dissolved in a mixed solution of pure water andacetonitrile (2:1) until the concentration is 1 mmol/500 mL, thusobtaining a linear crude peptide solution; Step 6, dilute ammonia wateris dropwisely added to the MRG linear crude peptide solution to adjustthe pH of the MRG linear crude peptide solution to 7.1-7.3, and stirredat a temperature of about 30° C. for cyclization experiment. Thecyclization time is 20-40 min; whether the cyclization is complete isdetected by high performance liquid chromatography (HPLC) analysis andthe cyclization is terminated by adding acetic acid to adjust pH to theacidic condition after the cyclization is complete; Step 7, the targetproduct crude product solution of step 6 is separated and purified byC₁₈ reversed-phase high performance liquid chromatography column, andthe high purity product is obtained after rotary evaporation andfreeze-drying.
 8. A method for treating a disease associated withStaphylococcus aureus infection comprising administering atherapeutically effective amount of the peptide of claim
 1. 9. Thepeptide of claim 1, wherein m=3-17.
 10. The peptide of claim 9, whereinm=6-14.
 11. The peptide of claim 10, wherein m=8-12.
 12. The peptide ofclaim 1, wherein X=CONH.
 13. The peptide of claim 1, wherein n=1-7. 14.The peptide of claim 13, wherein n=2-4.
 15. The peptide of claim 14,wherein n=3.
 16. The peptide of claim 1, wherein CH₃—(CH₂)m- isCH₃(CH₂)₁₀-(m=10), which forms a lauroyl modified G when being linked toX=CONH.